Gille J J, Wientjes N M, Lafleur M V, Joenje H, Retèl J
Department of Medical Oncology, Vrije Universiteit, Amsterdam, The Netherlands.
Carcinogenesis. 1996 Jan;17(1):5-11. doi: 10.1093/carcin/17.1.5.
In order to study the biological consequences of DNA damage induced by H2O2-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H2O2, Fe(3+)-citrate and ascorbate either alone or in combination. Induction of DNA lesions was determined as well as the biological activity of the phage DNA. Exposure to H2O2 alone resulted in max. 0.2 single-strand breaks per molecule; in the presence of Fe(3+)-citrate, the yield was approximately 4-fold higher. Under both conditions no double-strand breaks could be detected and the biological activity was not diminished. This indicates that low levels of single-strand breaks as generated by H2O2/Fe(3+)-citrate do not inactivate PM2 DNA. Exposure to ascorbate in the presence Fe(3+)-citrate resulted in extensive induction of single-strand breaks. However, at ascorbate concentration where approximately 3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished. At 5 mM ascorbate, single-strand breaks were above the detection limit. Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation was calculated to be approximately 3%. When PM2 DNA was exposed to H2O2 in the presence of ascorbate/Fe(3+)-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand breaks and biological inactivation, suggesting that one or more reactive species sensitive to H2O2 play a critical role. The .OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than .OH are involved. Our data suggest that other reactive species than .OH, such as the ferryl ion, are involved in H2O2-mediated DNA damage induction and biological inactivation.
为了研究由H2O2介导的自由基反应诱导的DNA损伤的生物学后果，将来自噬菌体PM2的DNA单独或组合暴露于H2O2、柠檬酸铁（Fe(3+)）和抗坏血酸。测定了DNA损伤的诱导情况以及噬菌体DNA的生物学活性。单独暴露于H2O2时，每个分子最多产生0.2个单链断裂；在柠檬酸铁（Fe(3+)）存在的情况下，产量约高4倍。在这两种情况下均未检测到双链断裂，并且生物学活性未降低。这表明H2O2/柠檬酸铁（Fe(3+)）产生的低水平单链断裂不会使PM2 DNA失活。在柠檬酸铁（Fe(3+)）存在下暴露于抗坏血酸会导致单链断裂大量诱导。然而，在每个分子诱导约3个单链断裂的抗坏血酸浓度下，同样未检测到双链断裂，并且DNA的生物学活性未降低。在5 mM抗坏血酸时，单链断裂超出检测限。在这些条件下，诱导了0.02个双链断裂，生物学活性降低至50%。计算得出双链断裂对生物学失活的贡献约为3%。当PM2 DNA在抗坏血酸/柠檬酸铁（Fe(3+)）存在下暴露于H2O2时，对于双链断裂的诱导和生物学失活均观察到典型的双相剂量效应关系，这表明一种或多种对H2O2敏感的活性物质起关键作用。羟基自由基（·OH）清除剂叔丁醇在保护PM2 DNA方面似乎相对无效，这可能表明涉及的活性物质不是羟基自由基（·OH）。我们的数据表明，除了羟基自由基（·OH）之外的其他活性物质，如高铁离子，参与了H2O2介导的DNA损伤诱导和生物学失活。
